Mutant Cystic Fibrosis Transmembrane Conductance Regulator Protein Research Articles

Rescue of Rare CFTR Trafficking Mutants Highlights a Structural Location-Dependent Pattern for Correction.

Cystic Fibrosis (CF) is a genetic disease caused by mutations in the gene encoding the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channel. Currently, more than 2100 variants have been identified in the gene, with a large number being very rare. The approval of modulators that act on mutant CFTR protein, correcting its molecular defect and thus alleviating the burden of the disease, revolutionized the field of CF. However, these drugs do not apply to all patients with CF, especially those with rare mutations-for which there is a lack of knowledge on the molecular mechanisms of the disease and the response to modulators. In this work, we evaluated the impact of several rare putative class II mutations on the expression, processing, and response of CFTR to modulators. Novel cell models consisting of bronchial epithelial cell lines expressing CFTR with 14 rare variants were created. The variants studied are localized at Transmembrane Domain 1 (TMD1) or very close to the signature motif of Nucleotide Binding Domain 1 (NBD1). Our data show that all mutations analyzed significantly decrease CFTR processing and while TMD1 mutations respond to modulators, those localized in NBD1 do not. Molecular modeling calculations confirm that the mutations in NBD1 induce greater destabilization of CFTR structure than those in TMD1. Furthermore, the structural proximity of TMD1 mutants to the reported binding site of CFTR modulators such as VX-809 and VX-661, make them more efficient in stabilizing the CFTR mutants analyzed. Overall, our data suggest a pattern for mutation location and impact in response to modulators that correlates with the global effect of the mutations on CFTR structure.

A Developmental Role of the Cystic Fibrosis Transmembrane Conductance Regulator in Cystic Fibrosis Lung Disease Pathogenesis.

The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein is a cAMP-activated anion channel that is critical for regulating fluid and ion transport across the epithelium. This process is disrupted in CF epithelia, and patients harbouring CF-causing mutations experience reduced lung function as a result, associated with the increased rate of mortality. Much progress has been made in CF research leading to treatments that improve CFTR function, including small molecule modulators. However, clinical outcomes are not necessarily mutation-specific as individuals harboring the same genetic mutation may present with varying disease manifestations and responses to therapy. This suggests that the CFTR protein may have alternative functions that remain under-appreciated and yet can impact disease. In this mini review, we highlight some notable research implicating an important role of CFTR protein during early lung development and how mutant CFTR proteins may impact CF airway disease pathogenesis. We also discuss recent novel cell and animal models that can now be used to identify a developmental cause of CF lung disease.

Lumacaftor and Matrine: Possible Therapeutic Combination to Counteract the Inflammatory Process in Cystic Fibrosis.

Cystic fibrosis is a monogenic, autosomal, recessive disease characterized by an alteration of chloride transport caused by mutations in the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) gene. The loss of Phe residue in position 508 (ΔF508-CFTR) causes an incorrect folding of the protein causing its degradation and electrolyte imbalance. CF patients are extremely predisposed to the development of a chronic inflammatory process of the bronchopulmonary system. When the cells of a tissue are damaged, the immune cells are activated and trigger the production of free radicals, provoking an inflammatory process. In addition to routine therapies, today drugs called correctors are available for mutations such as ΔF508-CFTR as well as for others less frequent ones. These active molecules are supposed to facilitate the maturation of the mutant CFTR protein, allowing it to reach the apical membrane of the epithelial cell. Matrine induces ΔF508-CFTR release from the endoplasmic reticulum to cell cytosol and its localization on the cell membrane. We now have evidence that Matrine and Lumacaftor not only restore the transport of mutant CFTR protein, but probably also counteract the inflammatory process by improving the course of the disease.

Role of Cystic Fibrosis Bronchial Epithelium in Neutrophil Chemotaxis.

A hallmark of cystic fibrosis (CF) chronic respiratory disease is an extensive neutrophil infiltrate in the mucosa filling the bronchial lumen, starting early in life for CF infants. The genetic defect of the CF Transmembrane conductance Regulator (CFTR) ion channel promotes dehydration of the airway surface liquid, alters mucus properties, and decreases mucociliary clearance, favoring the onset of recurrent and, ultimately, chronic bacterial infection. Neutrophil infiltrates are unable to clear bacterial infection and, as an adverse effect, contribute to mucosal tissue damage by releasing proteases and reactive oxygen species. Moreover, the rapid cellular turnover of lumenal neutrophils releases nucleic acids that further alter the mucus viscosity. A prominent role in the recruitment of neutrophil in bronchial mucosa is played by CF bronchial epithelial cells carrying the defective CFTR protein and are exposed to whole bacteria and bacterial products, making pharmacological approaches to regulate the exaggerated neutrophil chemotaxis in CF a relevant therapeutic target. Here we revise: (a) the major receptors, kinases, and transcription factors leading to the expression, and release of neutrophil chemokines in bronchial epithelial cells; (b) the role of intracellular calcium homeostasis and, in particular, the calcium crosstalk between endoplasmic reticulum and mitochondria; (c) the epigenetic regulation of the key chemokines; (d) the role of mutant CFTR protein as a co-regulator of chemokines together with the host-pathogen interactions; and (e) different pharmacological strategies to regulate the expression of chemokines in CF bronchial epithelial cells through novel drug discovery and drug repurposing.

Corticosteroid use and increased CXCR2 levels on leukocytes are associated with lumacaftor/ivacaftor discontinuation in cystic fibrosis patients homozygous for the F508del CFTR mutation.

Cystic fibrosis (CF) is the most common life-shortening genetic disease and is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Several current therapies aim at improving availability and/or function of the mutant CFTR proteins. The combination therapeutic lumacaftor/ivacaftor (Orkambi, luma/iva) partially corrects folding and potentiates CFTR function impaired by the F508del mutation. Despite the potential for clinical benefit, a substantial number of patients discontinue treatment due to intolerable adverse effects. The aim of the present study is to identify differences between individuals who continued treatment and those who discontinued due to adverse respiratory effects to potentially inform treatment decisions. Clinical data from the year prior to treatment initiation were analyzed from 82 patients homozygous for the F508del mutation treated at the Colorado Adult CF Program. Blood samples were collected from 30 of these subjects before initiation of treatment to examine expression of circulating leukocyte surface antigens and cytokines. Clinical and demographic characteristics were analyzed along with inflammatory markers to determine biomarkers of drug discontinuation. The use of oral prednisone and/or nasal budesonide in the year prior to luma/iva initiation was more prevalent in CF subjects who did not tolerate luma/iva (82% vs. 43%). Increased age, but not gender or initial lung function, was associated with higher probability of discontinuing treatment due to side effects overall. Worse lung function (lower ppFEV1, ppFEF25-75 ≤ 60%) was associated with higher incidence of discontinuing treatment due to pulmonary adverse effects. In a nested cohort of patients, increased surface levels of CXCR2 on CD14+CD16- monocytes were associated with discontinuation. Overall, the patients who tolerated luma/iva were distinguishable from those who did not tolerate the drug based on clinical and cellular markers obtained prior to treatment initiation.

Chronic β2AR stimulation limits CFTR activation in human airway epithelia.

Traditional pulmonary therapies for cystic fibrosis (CF) target the downstream effects of CF transmembrane conductance regulator (CFTR) dysfunction (the cause of CF). Use of one such therapy, β-adrenergic bronchodilators (such as albuterol), is nearly universal for airway clearance. Conversely, novel modulator therapies restore function to select mutant CFTR proteins, offering a disease-modifying treatment. Recent trials of modulators targeting F508del-CFTR, the most common CFTR mutation, suggest that chronic β-agonist use may undermine clinical modulator benefits. We therefore sought to understand the impact of chronic or excess β-agonist exposure on CFTR activation in human airway epithelium. The present studies demonstrate a greater than 60% reduction in both wild-type and modulator-corrected F508del-CFTR activation following chronic exposure to short- and long-acting β-agonists. This reduction was due to reduced cellular generation of cAMP downstream of the β-2 adrenergic receptor-G protein complex. Our results point towards a posttranscriptional reduction in adenylyl cyclase function as the mechanism of impaired CFTR activation produced by prolonged β-agonist exposure. β-Agonist-induced CFTR dysfunction was sufficient to abrogate VX809/VX770 modulation of F508del-CFTR in vitro. Understanding the clinical relevance of our observations is critical for CF patients using these drugs, and for investigators to inform future CFTR modulator drug trials.

Functional reconstitution and channel activity measurements of purified wildtype and mutant CFTR protein.

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Aggregates of mutant CFTR fragments in airway epithelial cells of CF lungs: New pathologic observations

Cystic fibrosis (CF) is caused by a mutation in the CF transmembrane conductance regulator (CFTR) gene resulting in a loss of Cl− channel function, disrupting ion and fluid homeostasis, leading to severe lung disease with airway obstruction due to mucus plugging and inflammation. The most common CFTR mutation, F508del, occurs in 90% of patients causing the mutant CFTR protein to misfold and trigger an endoplasmic reticulum based recycling response. Despite extensive research into the pathobiology of CF lung disease, little attention has been paid to the cellular changes accounting for the pathogenesis of CF lung disease. Here we report a novel finding of intracellular retention and accumulation of a cleaved fragment of F508del CFTR in concert with autophagic like phagolysosomes in the airway epithelium of patients with F508del CFTR. Aggregates consisting of poly-ubiquitinylated fragments of only the N-terminal domain of F508del CFTR but not the full-length molecule accumulate to appreciable levels. Importantly, these undegraded intracytoplasmic aggregates representing the NT-NBD1 domain of F508del CFTR were found in ciliated, in basal, and in pulmonary neuroendocrine cells. Aggregates were found in both native lung tissues and ex-vivo primary cultures of bronchial epithelial cells from CF donors, but not in normal control lungs. Our findings present a new, heretofore, unrecognized innate CF gene related cell defect and a potential contributing factor to the pathogenesis of CF lung disease. Mutant CFTR intracytoplasmic aggregates could be analogous to the accumulation of misfolded proteins in other degenerative disorders and in pulmonary “conformational protein-associated” diseases. Consequently, potential alterations to the functional integrity of airway epithelium and regenerative capacity may represent a critical new element in the pathogenesis of CF lung disease.

Ivacaftor treatment of cystic fibrosis patients with the G551D mutation: a review of the evidence

Cystic fibrosis (CF) is a recessive disorder caused by mutations in the gene that encodes the CF transmembrane conductance regulator (CFTR) protein. CFTR protein is a chloride and bicarbonate channel that is critical for normal epithelial ion transport and hydration of epithelial surfaces. Current CF care is supportive, but recent breakthroughs have occurred with the advent of novel therapeutic strategies that assist the function of mutant CFTR proteins. The development and key clinical trial results of ivacaftor, a small molecule that targets gating defects in disease-causing CFTR mutations including G551D CFTR, are summarized in this review. The G551D mutation is reasonably common in the CF patient population and produces a CFTR protein that localizes normally to the plasma membrane, but fails to open in response to cellular cues. Ivacaftor treatment produces dramatic improvements in lung function, weight, lung disease stability, patient-reported outcomes, and CFTR biomarkers in patients with CF harboring the G551D CFTR mutation compared with placebo controls and patients with two copies of the common F508del CFTR mutation. The unprecedented success of ivacaftor treatment for the G551D CF patient population has generated excitement in the CF care community regarding the expansion of its use to other CF patient populations with primary or secondary gating defects.

Novel opportunities for CFTR-targeting drug development using organoids.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR mutations lead to production of non-functional CFTR, reduced amounts of normal functioning CFTR or misfolded CFTR with defects in trafficking or function. For decades, CF treatment has been focused on the symptoms of CF, but pharmacotherapy using small molecules that target the basic defect of CF, the mutant CFTR protein, is now possible for a limited amount of subjects with CF. This raises the exciting possibility that the majority of people with CF may receive effective treatment targeting the different CFTR mutants in the future. We recently described a functional CFTR assay using rectal biopsies from subjects with CF that were cultured in vitro into self-organizing mini-guts or organoids. We here describe how this model may assist in the discovery of new CFTR-targeting drugs, the subjects that may benefit from these drugs, and the mechanisms underlying variability in CFTR genotype-phenotype relations.

Pharmacological Correctors of Mutant CFTR Mistrafficking

The lack of phenylalanine 508 (ΔF508 mutation) in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) Cl− channel represents the most frequent cause of CF, a genetic disease affecting multiple organs such as lung, pancreas, and liver. ΔF508 causes instability and misfolding of CFTR protein leading to early degradation in the endoplasmic reticulum and accelerated removal from the plasma membrane. Pharmacological correctors of mutant CFTR protein have been identified by high-throughput screening of large chemical libraries, by in silico docking of virtual compounds on CFTR structure models, or by using compounds that affect the whole proteome (e.g., histone deacetylase inhibitors) or a single CFTR-interacting protein. The presence of multiple defects of the CFTR protein caused by the ΔF508 mutation and the redundancy of quality control mechanisms detecting ΔF508-CFTR as a defective protein impose a ceiling to the maximal effect that a single compound (corrector) may obtain. Therefore, treatment of patients with the most frequent CF mutation may require the optimized combination of two drugs having additive or synergic effects.

Synthesis and Properties of Molecular Probes for the Rescue Site on Mutant Cystic Fibrosis Transmembrane Conductance Regulator

Cystic fibrosis is a genetic disease caused by mutations in the gene for the cystic fibrosis transmembrane conductance regulator (CFTR) protein. In vitro experiments have demonstrated that 4-methyl-2-(5-phenyl-1H-pyrazol-3-yl)phenol (VRT-532, 1) is able to partially restore the function of mutant CFTR proteins. To help elucidate the nature of the interactions between 1 and mutant CFTR, molecular probes based on the structure of 1 have been prepared. These include a photoreactive aryl azide derivative 11 and a fluorescent dansyl sulfonamide 15. Additionally, a method for hydrogen isotope exchange on 1 has been developed, which could be used for the incorporation of radioactive tritium. Using iodide efflux assays, the probe molecules have been demonstrated to modulate the activity of mutant CFTR in the same manner as 1. These probe molecules enable a number of biochemical experiments aimed at understanding how 1 rescues the function of mutant CFTR. This understanding can in turn aid in the design and development of more efficacious compounds which may serve as therapeutic agents in the treatment of cystic fibrosis.

Mutations that Disrupt Formation of Functional CFTR

Cystic fibrosis (CF) is caused by a loss-of-function of the cystic fibrosis transmembrane conductance regulator (CFTR) channel. Many mutations that cause CF, including the most common disease allele ΔF508, interfere with CFTR function because the mutant protein does not efficiently fold into the native channel structure. Significantly, when these mutant proteins are induced to fold, in experimental systems, some CFTR function is recovered, suggesting an avenue for therapeutic development. Exploiting this opportunity requires detailed knowledge of the basic processes of CFTR folding and the steps altered by the disease-causing mutations. Models of CFTR place the critical F508 residue on the surface of one of the two nucleotide binding domains (NBD1) at a predicted interface with the intracellular loop (ICL4) in the second of two transmembrane domains (TMD2). A variety of biophysical, biochemical, and cell biological studies demonstrate that CFTR folds in a hierarchical manner, with folding of the domains occurring first, during translation and, later, the partially folded domains associating to form the final, functional CFTR structure. Consistent with the location of F508 in the structural models, its deletion interferes with both the folding of NBD1 and with subsequent steps of domain-domain association. The detailed energetics and kinetics of these processes provide insight into the fundamental mechanisms by which integral membrane proteins achieve their native structures and reveal obstacles to and suggest strategies for improving the folding efficiency of the mutant CFTR protein. Supported by NIH-NIDDK, NIH-NIDCR, Reata Pharmaceuticals, CFF.

The Use of Small Molecules to Correct Defects in CFTR Folding, Maturation, and Channel Activity

Cystic Fibrosis, one of the most common inherited lethal disease among Caucasians, is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. The CFTR protein acts as a gated Cl- channel at the apical membrane of epithelial cells, thereby facilitating proper hydration of mucosal linings. Disease causing mutations in the CFTR protein can affect a variety of steps in the biogenesis of a functional protein including the folding and trafficking of CFTR as well as the channel activity of plasma membrane-localized protein. Therefore, current research is focused on the use of small molecules to not only correct folding defects but also to enhance channel activity of mutant CFTR proteins. This review discusses the current knowledge of the folding, trafficking, and gating defects caused by CFTR mutations, the manner by which these defects are monitored by the cell, as well as the strategies which are currently being utilized to develop and screen for small molecule therapeutics. Keywords: Cystic fibrosis, chaperones, ER Quality control, correctors, potentiators

Misassembled mutant ΔF508 CFTR in the distal secretory pathway alters cellular lipid trafficking

Most patients with cystic fibrosis (CF) have a single codon deletion (DeltaF508) in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) that impairs assembly of the multidomain glycoprotein. The mutant protein escapes endoplasmic reticulum (ER) quality control at low temperature, but is rapidly cleared from the distal secretory pathway and degraded in lysosomes. CF cells accumulate free cholesterol similar to Niemann-Pick disease type C cells. We show that this lipid alteration is caused by the presence of misassembled mutant CFTR proteins, including DeltaF508, in the distal secretory pathway rather than the absence of functional CFTR. By contrast, cholesterol distribution is not changed by either D572N CFTR, which does not mature even at low temperature, or G551D, which is processed normally but is inactive. On expression of the DeltaF508 mutant, cholesterol and glycosphingolipids accumulate in punctate endosomal structures and cholesterol esters are reduced, indicating a block in the translocation of cholesterol to the ER for esterification. This is overcome by Rab9 overexpression, resulting in clearance of accumulating intracellular cholesterol. Similar but less pronounced alterations in intracellular cholesterol distribution are observed on expression of a temperature-rescued mutant variant of the related ATP-binding cassette (ABC) protein multidrug resistance-associated protein 1 (MRP1). Thus, on escape from ER quality control, misassembled mutants of CFTR and MRP1 impair lipid homeostasis in endocytic compartments.

Exposure to sodium butyrate leads to functional downregulation of calcium-activated potassium channels in human airway epithelial cells

Cystic fibrosis (CF) is caused by genetic mutations that lead to dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. The most common mutation, DeltaF508, causes inefficient trafficking of mutant CFTR protein from the endoplasmic reticulum to the cell membrane. Therapeutic efforts have been aimed at increasing the level of DeltaF508-CFTR protein in the membrane using agents such as sodium butyrate. In this study, we investigated the effects of culturing a human airway epithelial cell line, Calu-3, in the presence of 5 mM sodium butyrate. Within 24 h, butyrate exposure caused a significant decrease in the basal, as well as Ca(2+)-activated, anion secretion by Calu-3 cell monolayers, determined by the change in transepithelial short-circuit current in response to the Ca(2+)-elevating agent thapsigargin. The secretory response to 1-ethyl-2-benzimidazolinone, an activator of the basolateral Ca(2+)-activated K(+) channel KCNN4, was similarly reduced by butyrate treatment. Quantitative PCR revealed that these functional effects were associated with dramatic decreases in mRNA for both KCNN4 and CFTR. Furthermore, the KCNQ1 K(+) channel was upregulated after butyrate treatment. We suggest that prolonged exposure to sodium butyrate downregulates the expression of both KCNN4 and CFTR, leading to a functional loss of Ca(2+)-activated anion secretion. Thus, butyrate may inhibit, rather than stimulate, the anion secretory capacity of human epithelial cells that express wild-type CFTR, particularly in tissues that normally exhibit robust Ca(2+)-activated secretion.

Dimeric cystic fibrosis transmembrane conductance regulator exists in the plasma membrane.

CFTR (cystic fibrosis transmembrane conductance regulator) mediates chloride conduction across the apical membrane of epithelia, and mutations in CFTR lead to defective epithelial fluid transport. Recently, there has been considerable interest in determining the quaternary structure of CFTR at the cell surface, as such information is a key to understand the molecular basis for pathogenesis in patients harbouring disease-causing mutations. In our previous work [Ramjeesingh, Li, Kogan, Wang, Huan and Bear (2001) Biochemistry 40, 10700-10706], we showed that monomeric CFTR is the minimal functional form of the protein, yet when expressed in Sf 9 cells using the baculovirus system, it also exists as dimers. The purpose of the present study was to determine if dimeric CFTR exists at the surface of mammalian cells, and particularly in epithelial cells. CFTR solubilized from membranes prepared from Chinese-hamster ovary cells stably expressing CFTR and from T84 epithelial cells migrates as predicted for monomeric, dimeric and larger complexes when subjected to sizing by gel filtration and analysis by non-dissociative electrophoresis. Purification of plasma membranes led to the enrichment of CFTR dimers and this structure exists as the complex glycosylated form of the protein, supporting the concept that dimeric CFTR is physiologically relevant. Consistent with its localization in plasma membranes, dimeric CFTR was labelled by surface biotinylation. Furthermore, dimeric CFTR was captured at the apical surface of intact epithelial cells by application of a membrane-impermeable chemical cross-linker. Therefore it follows from the present study that CFTR dimers exist at the surface of epithelial cells. Further studies are necessary to understand the impact of dimerization on the cell biology of wild-type and mutant CFTR proteins.

CFTR directly mediates nucleotide-regulated glutathione flux.

Studies have shown that expression of cystic fibrosis transmembrane conductance regulator (CFTR) is associated with enhanced glutathione (GSH) efflux from airway epithelial cells, implicating a role for CFTR in the control of oxidative stress in the airways. To define the mechanism underlying CFTR-associated GSH flux, we studied wild-type and mutant CFTR proteins expressed in Sf9 membranes, as well as purified and reconstituted CFTR. We show that CFTR-expressing membrane vesicles mediate nucleotide-activated GSH flux, which is disrupted in the R347D pore mutant, and in the Walker A K464A and K1250A mutants. Further, we reveal that purified CFTR protein alone directly mediates nucleotide-dependent GSH flux. Interestingly, although ATP supports GSH flux through CFTR, this activity is enhanced in the presence of the non-hydrolyzable ATP analog AMP-PNP. These findings corroborate previous suggestions that CFTR pore properties can vary with the nature of the nucleotide interaction. In conclusion, our data demonstrate that GSH flux is an intrinsic function of CFTR and prompt future examination of the role of this function in airway biology in health and disease.

Regulatory Interaction between the Cystic Fibrosis Transmembrane Conductance Regulator and HCO[formula omitted] Salvage Mechanisms in Model Systems and the Mouse Pancreatic Duct

The pancreatic duct expresses cystic fibrosis transmembrane conductance regulator (CFTR) and HCO3- secretory and salvage mechanisms in the luminal membrane. Although CFTR plays a prominent role in HCO3- secretion, the role of CFTR in HCO3- salvage is not known. In the present work, we used molecular, biochemical, and functional approaches to study the regulatory interaction between CFTR and the HCO3- salvage mechanism Na+/H+ exchanger isoform 3 (NHE3) in heterologous expression systems and in the native pancreatic duct. We found that CFTR regulates NHE3 activity by both acute and chronic mechanisms. In the pancreatic duct, CFTR increases expression of NHE3 in the luminal membrane. Thus, luminal expression of NHE3 was reduced by 53% in ducts of homozygote DeltaF508 mice. Accordingly, luminal Na+-dependent and HOE694- sensitive recovery from an acid load was reduced by 60% in ducts of DeltaF508 mice. CFTR and NHE3 were co-immunoprecipitated from PS120 cells expressing both proteins and the pancreatic duct of wild type mice but not from PS120 cells lacking CFTR or the pancreas of DeltaF508 mice. The interaction between CFTR and NHE3 required the COOH-terminal PDZ binding motif of CFTR, and mutant CFTR proteins lacking the C terminus were not co-immunoprecipitated with NHE3. Furthermore, when expressed in PS120 cells, wild type CFTR, but not CFTR mutants lacking the C-terminal PDZ binding motif, augmented cAMP-dependent inhibition of NHE3 activity by 31%. These findings reveal that CFTR controls overall HCO3- homeostasis by regulating both pancreatic ductal HCO3- secretory and salvage mechanisms.

Inflammation in cystic fibrosis airways: relationship to increased bacterial adherence.

It is unclear whether inflammation in the cystic fibrosis (CF) lung relates predominantly to bacterial infection, or occurs as a direct consequence of mutant cystic fibrosis transmembrane conductance regulator (CFTR) protein. Interleukin (IL)-8 secretion from CF and non-CF cell lines, and from CF and non-CF human primary nasal epithelial cells incubated with or without Pseudomonas aeruginosa, was measured. Activation of nuclear factor-kappaB (NF-kappaB) in unstimulated CF and non-CF nasal epithelial cells, cell lines and murine tissues was measured by gel-shift assays. No significant difference in basal IL-8 production or NF-kappaB activation was observed between CF and non-CF primary nasal cells. However, CF cells exhibited a significantly (p<0.01) increased IL-8 secretion following P. aeruginosa stimulation. Equalization of the increased P. aeruginosa adherence observed in CF cells, to non-CF levels, resulted in comparable IL-8 secretion. Further, IL-8 production did not differ with mutations which result in either correctly localized CFTR, or in partial/total mislocalization of this protein. Similar levels of NF-kappaB activation were observed in a number of organs of wildtype and CF mice. Finally, IL-8 secretion and NF-kappaB activity were not consistently increased in CF cell lines. Cos-7 cell transfection with plasmids expressing deltaF508 or G551D mutant CFTR protein resulted in increased activation of a p50-containing NF-kappaB complex, but IL-8 secretion was similar to wild-type cells. The authors conclude that the stimulus produced by Pseudomonas aeruginosa is the predominant inflammatory trigger in their models.